Mycobacterium strains with modified erp gene and vaccine composition containing same

ABSTRACT

Mycobacterium  strains in which the erp gene is modified, and vaccine compositions comprising such  Mycobacterium  strains are provided. The modification of the erp gene may decrease the virulence and the persistence of the  Mycobacterium  strains.

This application is a continuation of U.S. application Ser. No.09/463,194, filed Jan. 21, 2000 (now U.S. Pat. No. 6,585,976), which isa National Stage application of PCT/FR98/01627, filed Jul. 22, 1998, thedisclosures of both of which are hereby incorporated in their entiretiesby reference.

The invention relates to a Mycobacterium strain with modified erp geneand the vaccine composition containing same.

Tuberculosis is an infectious disease caused in most cases by inhalationof bacteria belonging to the complex of Mycobacterium tuberculosisspecies (M. africanum, M. bovis, M. tuberculosis). With eight millionnew human cases annually causing three million deaths worldwide,tuberculosis remains a major public health problem (Sudre et al., 1992).The discovery of effective antibiotics (streptomycin, isoniazide,rifampicin and the like) appeared to allow the eradication of thisdisease. However, it is estimated that currently only 50% of patientsare diagnosed and receive treatment. This treatment is ofteninappropriate or poorly monitored and leads to the appearance of anincreasing number of antibiotic-resistant and even polychemoresistantstrains (Dooley et al., 1992). In this context, the development of avaccinal prophylaxis appears as a preferred solution for the control anderadication of tuberculosis.

The fact that an attenuated pathogenic bacterium is used as a componentof a vaccine has been widely described and implemented in the prior art.The methods for obtaining such attenuated bacteria comprise the randomselection of mutants induced chemically or by irradiation, or theproduction of recombinant bacteria of pathogenic origin in which a geneinvolved in a metabolic pathway has been inactivated by geneticengineering.

Straley et al. (1984) have studied the survival of avirulent mutants ofYersinia pestis which are deficient in one or more metabolic pathways.

Noriega et al. (1994) have manufactured, by genetic engineering, an oralShigella strain intended to be used as a vaccine prototype byintroducing deletions into a gene (aroA) encoding a protein involved ina metabolic pathway for an aromatic amino acid and they havedemonstrated that the resulting defective recombinant Shigella strainswere capable of inducing protective antibodies against the wild-typepathogen.

A major study has also been carried out using Salmonella as a model. Seefor example the reports by Hoiseth et al. (1981), Levine et al. (1987),Oyston et al. (1995) and Curtiss (1990).

However, similar studies have not yet been carried out for Mycobacteriumtuberculosis, the etiological agent of tuberculosis (TB), which infectsa third of the world population and kills three million people per year.Tuberculosis is the most important cause of mortality in the worldcaused by a group of infectious organisms (Bloom and Murray, 1992)grouped under the name “M. tuberculosis complex”. According to the WHO,more people died of tuberculosis in 1995 than during any previous year.It has been estimated that up to half a billion people will suffer fromtuberculosis in the next 50 years. However, in spite of its importance,the genetic determinants of the virulence and persistance of M.tuberculosis remain scarcely characterized.

Indeed, the virulence of pathogenic mycobacteria is associated withtheir ability to grow and persist at the intracellular level. Bacteriaof the M. tuberculosis complex parasitize the phagocytic cells in whichthey live and multiply in a specialized vacuolar compartment called thephagosome. The phagosomes containing live M. tuberculosis do not acidifyand escape fusion with the lysosomes. The mechanisms by which M.tuberculosis make their phagosome more hospitable remain unknown and themycobacterial genes affecting their intracellular growth andmultiplication are being actively investigated.

The extreme difficulty of creating defined mutants of M. tuberculosis,either by allelic exchange or by transposon mutagenesis, has preventedthe identification of these virulence factors according to thepostulates of Koch (Falkow, 1988; Jacobs, 1992). Alternative geneticstrategies have been used instead, including the complementation of anon-pathogenic bacterium (Arruda et al., 1993) and of spontaneousavirulent mutants with virulent M. tuberculosis (Pascopella et al.,1994) and virulent M. bovis (Collins et al., 1995) chromosomal DNAlibraries. Although these studies have identified genes potentiallyinvolved in the entry into the epithelial cells and conferring a growthadvantage in vivo, the great majority of the mycobacterial genesinvolved in the virulence and survival in the host organism remainunknown. The development of effective mutagene systems is therefore thepriority for mycobacterial genetics.

One method for the creation of mutants is allelic exchange mutagenesis.Recently, allelic exchanges taking place with a low frequency have beendemonstrated in bacteria of the M. tuberculosis complex using a suicidevector (Reyrat et al., 1995; Azad et al., 1996) and novel protocolsallowing easier detection of the allelic exchange mutants have also beendeveloped (Norman et al., 1995; Balasubramamian et al., 1996; Pelicic etal., FEMS Microbiol. Lett. 1996). However, the detection of a very rareallelic exchange event is prevented by low transformation efficienciesand the high frequency of illegitimate recombinations. Thus, manyMycobacterium genes still remain refractory to allelic exchange by meansof the available technologies.

More particularly, the allelic exchange mutagenesis systems require theuse of more efficient methods. The problems encountered may be overcomeby the use of a replicative vector which is effectively conditionallylost. The possibility of introducing such vectors makes it possible toavoid the problems resulting from the low transformation efficiencies.Thus, under counterselection conditions, the clones still containing thevector are eliminated, thus allowing the detection of very rare geneticevents. Such a system has been recently developed. Using a replicativevector under certain conditions which is lost at 39° C. in M. smegmatis,the first library of mycobacterial insertion mutants was constructed inthis rapidly growing model strain (Guilhot et al., 1994). However, theheat-sensitive vectors used are only slightly heat-sensitive inslow-growing mycobacteria of the M. tuberculosis complex and thereforecannot be used in these species for allelic exchange mutagenesis(unpublished data).

The inventors have succeeded in altering the virulence and thepersistance of Mycobacterium strains in the host cells.

They have indeed produced a Mycobacterium strain one gene of which hasbeen modified so as to attenuate its virulence.

Modified gene is understood to mean a gene which has undergone amodification abolishing the production of the corresponding protein orallowing the production of a protein which is at least 20% different interms of activity compared with the natural protein.

BCG (Bacille Calmette-Guérin), an avirulent strain derived from M.bovis, is widely used worldwide as a vaccine against tuberculosis.However, while BCG can be administered without any problem toindividuals with no immune deficiency, the same is not true forimmunosuppressed individuals such as people infected with the AIDSvirus, people who have had a marrow transplant, people suffering from acancer, or people with altered functioning of one of the components ofthe immune system.

That is the reason why the present invention relates to a Mycobacteriumstrain with limited persistance.

The gene modified in the Mycobacterium strain in accordance with theinvention is the erp gene. It may also be a gene having acomplementation homology (of at least 80%) with the erp gene.

Analysis of the deduced protein sequence of the erp gene shows that thelatter encodes a protein whose calculated molecular mass is 28 kDa. Thepresence of a signal sequence for export at an N-terminal position aswell as the existence of a C-terminal hydrophobic region suggest thatthe molecule can be anchored in the plasma membrane or located at thesurface of the bacilli. Furthermore, the central region of the proteincomprises two repeat regions each composed of 6 copies of a P(G/A)LTS(SEQ ID NO 1) motif positioned in tandem. This organization is similarto that found in many surface proteins associated with peptidoglycan inGram-positive bacteria and in Plasmodium.

A genetic methodology allowing the selection and the identification ofDNA fragments encoding exported proteins has recently been adapted forM. tuberculosis in the laboratory. This system is based on theproduction of libraries of M. tuberculosis DNA fragments fused with theE. coli alkaline phosphatase (phoA) gene lacking expression and exportsignals. Alkaline phosphatase (PhoA) possesses detectable enzymaticactivity only after export across the plasma membrane. Using thissystem, several DNA fragments allowing the export of PhoA inmycobacteria have been selected in the presence of a chromogenicsubstrate, and partially sequenced. One of the fusions carried by therecombinant plasmid pExp53 exhibits sequence similarities with an M.leprae gene which encodes a protein of 28 kDa, potentially located atthe surface of the bacillus. Furthermore, this protein is a major M.leprae antigen recognized by the sera of lepromatous leprosy patients(WO 9607745). We have furthermore determined, by molecular hybridizationexperiments, that the erp gene is unique in the M. tuberculosis genomeand that it is also present in the genome of the other members of thiscomplex of species (M. africanum, M. bovis, M. bovis BCG).

To allow the study of ERP and to confirm its localization at thesurface, specific anti-ERP antibodies were produced. For that, the ERPprotein fused with the maltose-binding protein (MalE/MBP) or fused witha C-terminal peptide containing 6 histidine residues (SEQ ID NO 2) wasproduced. This strategy made it possible to obtain, in a large quantity,recombinant ERP-MalE and ERP(His) 6 proteins expressed in Escherichiacoli. The purification of these molecules was carried out using thetechniques of affinity chromatography on a resin of amylose (MalEsystem) or of chelated nickel (Histidine system). The relative molecularweight, determined by SDS-PAGE electrophoresis, is 36 K. The differencewith the theoretical molecular weight may be attributed to a delay inelectrophoretic migration due to the high content (15%) of prolineresidues. A protocol for immunizing rabbits with the aid of the purifiedERP-MalE and ERP(His)6 chimeras made it possible to obtain polyclonalsera at a high titer which allow the specific detection of the ERPprotein.

With the aid of the antisera obtained in rabbits, the localization ofthe ERP protein in Mycobacterium tuberculosis was specified. Electronmicroscopy observations after immunolabeling with colloidal gold made itpossible to detect the presence of the ERP protein at the surface oftubercle bacilli derived from an in vitro culture. Thus, the ERP proteinis capable of exhibiting at the surface of the mycobacteria epitopes ofother antigens and for vaccinal or therapeutic purposes. Furthermore,similar experiments have made it possible to detect the ERP protein inmurine macrophages infected with M. tuberculosis.

To analyze the function of the ERP protein, a BCG strain in which theerp gene was modified by allelic exchange was constructed. The survivalof this strain in comparison with the wild-type strain was analyzed inthe mouse model. It was demonstrated that the mutation of the erp geneseverely affects the persistance of M. bovis BCG. This reduction inpersistance is observed in all the organs tested (spleen, liver, lungs).In addition to the role of the gene in the BCG survival process, theseobservations mean that the erp gene is expressed during the growth phasein vivo in the host.

More particularly, the modification of the erp gene is carried out bymutation, insertion, deletion or substitution; the modification of atleast one base pair is sufficient.

According to an advantageous embodiment of the strain in accordance withthe invention, the erp gene is modified by insertion of a nucleotide orpolynucleotide which may be a selectable gene. This gene may inparticular encode the resistance to an antibiotic such as kanamycin,spectinomycin or hygromycin.

The preferred Mycobacterium strains are those belonging to theMycobacterium genus, preferably to the Mycobacterium tuberculosiscomplex and still more preferably to the Mycobacterium tuberculosisspecies or to the Mycobacterium bovis species.

The present invention relates more particularly to the BOG erp::Knstrain also called BCG erp::aph (Collection Nationale de Cultures deMicroorganismes (“CNCM”), located at Institut Pasteur, 28, Rue duDocteur Roux, F-75724 PARIS CEDEX 15, France, Deposit No. I-1896, madeJul. 15, 1997) or a variant incapable of expressing the product of theactive erp gene as well as the M. tuberculosis H37Rv erp::aph strain(CNCM Deposit No. 1-2048, made jun. 29, 1998) or a variant incapable ofexpressing the gene product.

The invention also relates to a Mycobacterium strain whose erp gene ismodified and which is capable of producing, following recombinationevents, epitopes or antigenic determinants capable of immunizing and/orprotecting against pathogenic agents such as infectious agents or cancergenes, or of producing molecules leading to a modulation of the immunesystem such as cytokines, chemokines, soluble receptors for moleculesinteracting with agents leading to a pathological condition or inducersof immune responses such as IL2, IL4, IL10 or IL12 (in humans oranimals).

The present invention therefore also relates to a Mycobacterium strainas described above which is capable, in addition, of expressing apolynucleotide encoding a mycobacterium antigen of a species other thanthat to which said strain belongs, it being possible for thepolynucleotide in question to be foreign to the Mycobacterium genus.

A subject of the invention is also a purified polynucleotide comprisinga modified erp gene and a fragment of at least 60 nucleotidescorresponding to the whole or part of a gene encoding an exportedantigen of the Mycobacterium genus or encoding an antigen foreign to theMycobacterium genus.

The modification of the erp gene may be obtained, for example, byaddition, insertion or modification of nucleotides. In the context ofthe invention, the selection of the Mycobacterium strain whose erp geneis thus modified may be carried out by gene amplification and nucleotidesequencing or RFLP of the nucleic region mutated in said strain isolatedon agar according to the counterselection protocol in the presence ofsucrose (Pelicic et al., 1996), for example. An alternative consists incarrying out hybridizations under high stringency conditions (Berthet etal., 1995) characterized by the use of a probe corresponding to thewhole or part of the erp gene which has been genetically modified butwhich conserved at least 20% of its activity and which preferablyhybridizes with the whole or part of the modified gene present in thedesired strain.

The modification of the erp gene may also be carried out by means of arecombinant vector comprising the inactivated erp gene. This vector isused for the transformation of a Mycobacterium strain and should allowan allelic exchange with the wild-type erp gene with the aim ofmodifying it.

Advantageously, the vector in accordance with the invention comprises areplication origin which is heat-sensitive in mycobacteria. It may alsocomprise the counterselectable sacB gene optionally with a gene allowingpositive selection such as a gene encoding resistance to an antibiotic.

The modification of the erp gene in the vector in accordance with theinvention may be carried out as described above.

More particularly, said vector corresponds to the recombinant plasmidpIPX56 (CNCM Deposit No. I-1895, made Jul. 15, 1997). Indeed, thisplasmid consists of an E. coli—mycobacteria shuttle cloning vector ofthe pPR27 type (CNCM Deposit No. I-1730, made Jun. 19, 1996) comprisinga replication origin which is heat-sensitive in mycobacteria, thecounterselectable sacB gene and conferring resistance to gentamycin. Inthe plasmid pIPX56, an insertion of 5.1 kb of a PstI fragment wascarried out at the level of the unique PstI site of pPR27. This 5.1 kbfragment corresponds to a 3.9 kb DNA fragment of M. tuberculosiscomprising the erp gene, into which a cassette (1.2 kb) conferringresistance to kanamycin has been inserted. This plasmid therefore makesit possible to carry out allelic exchange experiments at the level ofthe erp locus in mycobacteria.

In the context of the present invention, it is also advantageous to beable to have a vector derived from pIPX56 comprising the unmodified erpgene.

The subject of the invention is therefore also the use of a recombinantvector as described above for the preparation of a Mycobacterium strainin accordance with the invention by allelic exchange.

Another subject of the invention is a method for the production of aMycobacterium strain as described above comprising the steps of:

-   -   transforming, with a vector as described above, a Mycobacterium        strain propagated at a permissive temperature,    -   culturing the colonies resulting from the transformation on a        medium supplemented with a selectable product and sucrose,    -   isolating the recombinant strain.

According to an advantageous embodiment of the method in accordance withthe invention, the selectable product is an antibiotic such askanamycin, spectinomycin or hygromycin.

By way of example, a recombinant Mycobacterium strain is produced inaccordance with the invention as follows:

-   -   a) the plasmid pIPX56 is introduced by electroporation into a        strain of the Mycobacterium tuberculosis complex propagated at a        permissive temperature (32° C.). This step makes it possible to        have a population of bacteria in which each individual possesses        several copies of the erp::Kn cassette;    -   b) a colony resulting from the transformation is cultured in        liquid medium at 32° C. for 10 days, and then the culture is        inoculated on plates containing kanamycin (50 mg/ml) and 2%        sucrose (weight/vol.) which are incubated at a nonpermissive        temperature at 39° C. This step makes it possible to enrich in        double homologous recombination events by counterselection and        elimination of the integrations of vectors (single homologous        recombination or illegitimate recombination).

The invention also relates to an immunogenic composition comprising aMycobacterium strain in accordance with the invention or obtained bycarrying out the method mentioned above.

It also relates to a vaccine composition comprising a Mycobacteriumstrain in accordance with the invention or obtained by carrying out themethod mentioned above, in combination with at least onepharmaceutically compatible excipient.

This vaccine composition is intended for the immunization of humans andanimals against a pathogenic strain of mycobacteria and comprises animmunogenic composition as described above in combination with apharmaceutically compatible excipient (such as a saline buffer),optionally in combination with at least one immunity adjuvant such asaluminum hydroxide or a compound belonging to the muramyl peptidefamily.

To obtain an adjuvant effect for the vaccine, many methods envisage theuse of agents such as aluminum hydroxide or phosphate (alum) which arecommonly used as a solution titrating 0.05 to 0.01% in aphosphate-buffered saline, mixed with synthetic polymers of sugar(Carbopol) as a 0.25% solution. Another suitable adjuvant compound isDDA (2 dimethyldioctadecylammonium bromide), as well as immunomodulatorysubstances such as the lymphokines (for example gamma-IFN, IL-1, IL-2and IL-12) or also gamma-IFN-inducing compounds such as poly I:C.

The vaccine composition in accordance with the present invention isadvantageously prepared in injectable form, for administration orally orby inhalation, or in liquid solution or in suspension; suitable solidforms intended to be prepared in solution or in liquid suspension beforeinjection can also be prepared.

Furthermore, the vaccine composition may contain minor components of anauxiliary substance such as wetting or emulsifying agents, agents forbuffering the pH or adjuvants which stimulate the efficacy of thevaccines.

The vaccine compositions of the invention are administered in a mannercompatible with the dosage formulation and in a therapeuticallyeffective and immunogenic quantity. The quantity to be administereddepends on the subject to be treated, including, for example, theindividual capacity of their immune system to induce an immune response.

The vaccine dosage will depend on the route of administration and willvary according to the age of the patient to be vaccinated and, to alesser degree, the size of this patient. Preferably, the vaccinecomposition according to the present invention is administered by theintradermal route in a single portion or by the oral route or byaerosol.

In some cases, it will be necessary to carry out multipleadministrations of the vaccine composition in accordance with thepresent invention without, however, generally exceeding sixadministrations, preferably four vaccinations. The successiveadministrations will normally be made at an interval of 2 to 12 weeks,preferably of 3 to 5 weeks. Periodic boosters at intervals of 1 to 5years, preferably 3 years, are desirable in order to maintain thedesired level of protective immunity.

The invention also relates to a diagnostic method which makes itpossible to discriminate between individuals, on the one hand, who havebeen vaccinated with the aid of a Mycobacterium strain no longerproducing active ERP and, on the other hand, those who have had anatural infection or a vaccination with the aid of a strain producingthe natural ERP protein.

Indeed, the individuals who have had a vaccination with a Mycobacteriumstrain no longer producing the natural ERP protein can be distinguishedby the absence, from a biological sample such as for example serum, ofantibodies directed against ERP and/or by the absence of T reactivity(measured for example during a test of proliferation or a test ofsecretion of cytokines or CTL test) against the purified ERP protein. Analternative also consists in testing for a differential reactivity withthe aid of antibodies directed against the unmodified part of thenatural ERP protein compared with the corresponding part of the mutatedERP protein.

The subject of the present invention is therefore also a method ofscreening individuals, to whom a vaccine composition in accordance withthe invention has been administered, comprising detecting the absence,from a biological sample from said individuals, of antibodies or of Tcells directed against the whole or part of the purified ERP protein, itbeing possible for the biological sample to be blood.

The subject of the invention is also a composition comprising themodified ERP protein.

Another aspect of the present invention relates to the repeat sequencespresent in the erp gene in particular of the strains of the M.tuberculosis complex. Indeed, in the majority of the cases studied bythe Inventors, the tuberculosis patients did not develop a humoralresponse against erp. The mice vaccinated with BCG do not develop ahumoral response against erp either. By contrast, the leprosy patientsdevelop a strong response against erp. The major difference between theERP protein from M. tuberculosis and the similar protein from M. lepraelies in the absence of repeats in M. leprae.

Consequently, the repeats may be responsible for the blocking of thehumoral response specifically against erp or even, in general, againstother antigens. It is indeed known that tuberculosis patients developonly a weak humoral response at the beginning of the tuberculosisdisease. It could therefore be possible to use the repeats carried byerp to inhibit the development of a specific humoral response bycombining these repeats with any antigen against which it is desired toavoid a humoral response being induced or perhaps even used by theserepeats to inhibit any humoral response in some advantageous contexts.This type of strategy could be appropriate for the following situations:avoiding the development of the humoral response against viral vaccinevectors (see table).

Thus, the present invention relates to the use of the repeat sequencesof the erp gene, optionally in combination with at least one otherantigen, for inhibiting the development of a humoral response.

It also relates to a vector for expression in a microorganism,characterized in that it comprises a nucleotide sequence encoding theERP protein lacking its repeat sequences. The microorganism harboringthe expression vector may be, for example, E. coli or any other organismwhich may be suitable for the expression of a nucleotide sequenceencoding the ERP protein lacking its repeat sequences, including themycobacteria.

The subject of the present invention is also a strain of mycobacteria,characterized in that the erp gene lacks its repeat sequences. Indeed,such a strain, having ERP with no repeats, would be immunogenic whilehaving a protective effect.

Furthermore, the subject of the present invention is a purifiedrecombinant ERP protein preferably produced by E. coli. Advantageously,this recombinant protein comprises six histidine residues at itsC-terminal end.

FIG. 1 represents the production of a BCG strain whose erp gene wasinactivated by insertion of a cassette for resistance to kanamycin.

FIG. 2 represents the number of cfu persisting in each of the relevantorgans as a function of the number of days following the intravenousinjection of either the wild-type BCG or the mutant BCG (BCG erp::Knalso called BCG erp::aph).

FIG. 3 represents the number of cfu resulting from the multiplication ofthe parental BCG (1173P2) and of the mutated BCG (erp::aph) in culturesof macrophages derived from BALB/C mouse medullary precursors.

FIG. 4 represents the number of cfu resulting from the multiplication ofthe parental (wt), mutated (ERP⁻) and complemented strains of BCG andH37Rv in cultures of macrophages derived from Balb/c mouse medullaryprecursors.

FIG. 5 represents the comparison of the number of cfu resulting from themultiplication of the parental, mutated and complemented BCG, on the onehand, with that of the parental, mutated and complemented H37Rv, on theother hand, in cultures of macrophages derived from Balb/c mousemedullary precursors, as a function of the organs (lungs, spleen orliver).

The invention is not limited to the above description and will beunderstood more clearly in the light of the examples.

Materials and Methods

Production and Purification of Recombinant ERP Protein

The region encoding ERP deprived of its signal sequence was amplified byPCR by means of the oligonucleotide primers His-2(5′-AAGGAGATCTTGTGCATATTTTCTTGTCTAC-3′) (SEQ ID: 3), and His-3(5′-AAGGAGATCTGGCGACCGGCACGGTGATTGG-3′) (SEQ ID: 4); digested with BglIIand cloned into the BamHI site of the expression plasmid pQE70 (QIAGENGmbH, Hilden, Germany). The resulting plasmid, designated pHis233, wassubjected to electroporation into the Escherichia coli M15 strain.

Two liters of cultures of the Escherichia coli M15 strain (pHis233) weregrown in a Luria-Bertani broth, induced with ITPG and were treated forthe purpose of protein purification under denaturing conditions using anickel-nitrilotriacetic acid (NTA) agarose resin as described by thesupplier (QIAGEN GmbH). Eluted ERP-His6 was dialyzed twice for 12 hourswith PBS and stored in the cold at −20° C. Two rabbits (New Zealandstrain) were immunized with 100 μg of protein and then every fifteendays with 150, 200 and 250 μg of ERP-His6 emulsified in incompleteFreund's adjuvant. The hyperimmune anti-ERP sera were obtained bybleeding the animals six weeks after immunization. The separation bySDS-PAGE electrophoresis and immunoabsorption were carried out asdescribed in J. Sambrook et al., 1987.

Immunocytochemistry (Full Setup)

Cells were fixed with a 0.1 M buffer of paraformaldehyde at 1%, washedin the same buffer and then applied to a nickel grid coated with Formvarcarbon, previously made hydrophilic by the “glow discharge” electricalprocess. The grids were then prepared by immunocytochemistry, rinsedwith distilled water and negatively stained with 1% ammonium molybdatein water.

Cryosections

The bacteria or infected macrophages (m.o.i.=1) were fixed with 2%paraformaldehyde and 0.2% glutaraldehyde in 0.1 M phosphate buffer. Thecells were harvested and entrapped in gelatin at 10%. The agglomeratedcells were incubated from two hours to a whole night in 1.8 M sucroseand polyvinylpyrrolidone at 15% (MW 10,000). Small blocks were mountedon “object holders”, cooled in liquid nitrogen and cryosectioned at−120° C. with a Reickert FCS cryo-ultamicrotome. Thin sections were thenrecovered in a drop of 2.3 M sucrose and applied to Formvarcarbon-coated nickel grids. The grids were then treated forimmunocytochemistry, then rinsed with distilled water and included inmethyl cellulose containing 0.3% uranyl acetate.

Immunocytochemistry

Grids were treated with drops of the following reagents: NH₄Cl (50 mM)in PBS, 10 minutes, Bovine Serum Albumin (BSA) 1% (w/v) in PBS, 5minutes, an anti-ERP antiserum diluted 1/100 in PBS-BSA, 1 hour, PBS-BSA(three washes of 2 to 5 minutes each), conjugated with gold anti-rabbitIgG antibody (H+L chains) (grains of 10 nm or 5 nm in size, BritishBiocell International, UK) diluted 1/20 in gelatin from fish skinPBS-0.1% (Sigma), 30 to 45 minutes, PBS (one wash, 1 minute) anddistilled water (three washes of one minute each). The examples werethen fixed with 1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4)for 2 minutes.

Inactivation of the erp Gene

A DNA fragment of 3.9 kb comprising the full length of the erp gene wascut from pIX412 by a PstI digestion and cloned into the correspondingsite of pACYC177. The resulting plasmid, designated pPB1, was linearizedwith EcoRI which cuts at a unique site inside erp. In parallel, an aphcassette conferring resistance to kanamycin was cut by PstI digestion inthe plasmid pUC-4K. pPB1 and the aph fragment were cut with T4 DNApolymerase (Boehringer Mannheim) as recommended by the manufacturer, andligated together to give pPB2. A DNA fragment of 5.2 kb containingerp::aph was cut from pPB2 by PstI digestion and cloned intononreplicative pJQ200 giving rise to the vector pPB3. Five μg of pPB3were subjected to electroporation into M. bovis BCG which was thenplated on Middelbrook 7H11 plates supplemented with kanamycin (20μg/ml). Colonies were sorted by PCR with oligonucleotides flanking theEcoRI sites used for the insertion of aph and transferred (replicaspotting) to plates containing kanamycin (20 g/ml) and 2% sucrose. Aclone containing an insert of 1.3 kb and more sensitive to sucrose wasanalyzed by Southern blotting as described in Berthet et al., 1995.

Mice were kept and handled according to the directives of InstitutPasteur for the breeding of laboratory animals. The macrophages derivedfrom the spinal cord were isolated from seven-week old femurs, fromfemale BALB/c mice. The cells were inoculated at 5×10⁵ cells per wellinto Labtek™ 8-well culture chambers (Nunc) and were cultured for sevendays as described in Chastellier et al., 1995.

The counting of the CFUs was carried out as described in Lagranderie etal., 1996.

EXAMPLE 1 Generation of Anti-ERP Rabbit Polyclonal Sera

Four female New Zealand rabbits were immunized the first time with 100μg of ERP(his)6 protein. Secondary immunizations (boosters) wereperformed every 15 days with 150, 200 and then 250 μg of purifiedprotein. From two months after the first immunization, the seracollected had a sufficient titer to be used for the immunodetection inWestern blotting and by immunohistochemistry in electron microscopy. Forthe electron microscopy immunohistochemistry experiments, the sera werepurified by adsorption/elution on nitrocellulose strips containing ERP.

EXAMPLE 2 Construction of a BCG erp::Kn Strain

A M. tuberculosis DNA fragment of 3.9 kbp containing the erp gene wasobtained by PstI digestion of the plasmid pIPX412 (CNCM No. I-1463)(Berthet et al., 1995). This fragment was introduced into the cloningvector pACYC177, giving rise to the plasmid pPB1. The plasmid pPB1 wasthen linearized with the restriction enzyme EcoRI, cutting once at thelevel of a site situated in the erp gene. In parallel, a geneticcassette (aph) which is small in size (1.3 kbp), conferring theresistance to the antibiotic kanamycin, was prepared by restricting theplasmid pUC-4K with PstI. The pACYC177/EcoRI and aph/PstI fragments weretreated, under the conditions described by the supplier, with the Klenowfragment of DNA polymerase I of Escherichia coli and with the T4 phagepolymerase, respectively, in order to generate fragments with bluntends. These two fragments were then ligated and transformed in E. coli.A recombinant plasmid having inserted the aph cassette at the level ofthe erp gene was selected by colony hybridization and called pPB2. Thefragment containing erp::aph was then excized from pPB2 by PstIdigestion and introduced into the vector pJQ200 possibly allowingcounterselection in the presence of sucrose. The resulting plasmid wascalled pPB3. Plasmid pPB3 (three micrograms) was introduced into theMycobacterium bovis BCG Pasteur 1173 P2 strain by electroporation (Genepulser BioRad, 2500V, 200Ω, 25 μF). The transformed cells were incubatedfor 24 hours at 37° C. in 7H9 medium (5 ml) and then plated on 7H11plates containing kanamycin (20 μg/ml). The plates were incubated for 25days at 37° C. and thirty colonies of bacteria resistant to kanamycinwere subcloned individually both by PCR using a pair of oligonucleotidesflanking the EcoRI site of the erp gene, and by Southern blotting usingan internal erp probe. A recombinant clone, called BCG erp::Kn, wasselected on the following criteria:

1—by PCR, disappearance of the band corresponding to the wild-typeallele (500 bp) for the mutant BCG erp::Kn. Production of a PCRamplification fragment of 1800 bp (500+1300) signing the insertion ofthe aph cassette into the genomic copy of the erp gene;

2—by Southern blotting, detection of a signal for hybridization at 5.2kbp with the DNA of BCG 13K instead of 3.9 kbp for the wild-type BCG.Loss of the EcoRI site internal to the erp::aph gene.

EXAMPLE 3 Test of the Persistence of BCG erp::aph in Mice

A bacterial stock stored at −70° C. of the BCG erp::Kn strain wasproduced in the following manner: a colony grown on 7H11+Kn (20 μg/ml)was inoculated on potato/Sauton medium in the presence of kanamycin (20μg/ml) until a film is formed. This film will be used to produce aninoculum (10 μg/ml) for flasks containing liquid Sauton medium. After 8days of growth, the film thus formed in Sauton is recovered, ground andis resuspended in Beck-Proskauer medium supplemented with 6% glycerol(Vol./Vol.). The stock thus obtained titrated 4.8×10⁸ colony formingunits (CFU)/ml.

With the aid of this stock, BALB/c mice were injected intravenously with10⁶ cfu of wild-type BCG and mutant BCG (BCG erp::Kn) in suspension inPBS. Three organs, the spleen, the liver and the lungs, were removed ina sterile manner at days 1, 7, 14, 28, 42, 56 and 70. At each point, theorgans were ground in Beck-Proskauer medium and the bacteria wereinoculated at different dilutions on 7H11 plates with or withoutkanamycin (20 μg/ml). The number of viable bacteria present in thedifferent organs as a function of time was determined by counting theCFUs (FIG. 2).

EXAMPLE 4 Study of the Multiplication of BCG erp::Kn in the MacrophagesDerived from Mouse Medullary Precursors

Previous experiments have shown that the BCG erp::Kn strain no longerpersists in mice. The organ in which the elimination of BCG erp::Kn ismost marked is the lung. The alveolar macrophages represent the primarytarget of infection by the mycobacteria of the M. tuberculosis complex.To specify the cell type in which the persistence of BCG erp::Kn isaffected, we studied the multiplication of this strain in macrophagesderived from mouse medullary precursors (BMDP). For that, BMDPs derivedfrom the femur of 7-week old female BALB/CBYJICO mice were isolated andcultured (DMEM medium (Gibco BRL), glutamine 2 mM, fetal calf serum(Dominique Deutscher SA) 10% Vol./Vol., supernatant of L229 cells 10%)in 8-well Labtek™ chambers, at a cell density of 5×10⁴ cells in 400 μlof medium. The cells were cultured for 7 days before being infected forfour hours with BCG 1173 P2 (parental strain) or BCG erp::Kn (mutatedstrain) at a multiplicity of infection of 1. At various timespost-infection (Day 0, Day 1, Day 5, Day 12, Day 17), the infectedmacrophages were lyzed in a buffer preserving the integrity of themycobacteria and the lysate obtained was plated on dishes of 7H11 mediumat various dilutions (from 10⁰ to 10⁻⁶). The Petri dishes were incubatedat 37° C. for one month in order to measure the variation of the numberof colony forming units represented in FIG. 3.

EXAMPLE 5 Study of the Delayed Hypersensitivity Reaction Induced inGuinea Pigs by BCG erp::Kn

Delayed hypersensitivity reaction (DTH) reflects the induction of immuneresponses directed against mycobacterial components. An indurationhaving a diameter greater than a threshold value, caused by theintradermal injection of tuberculin, signs a prior contact withmycobacteria. This test allows a rapid diagnosis of the tuberculosisinfection in non-vaccinated subjects. However, this test is difficult touse in subjects vaccinated with BCG who are positive in this case. Wetested the capacity of BCG erp::Kn to induce a DTH reaction. For that,two groups of 5 guinea pigs (300 g males, Dunkin Hartley strain) wereimmunized with 5×10⁵ viable units of BCG 1173P2 or erp::Kn respectively.One month later, the same animals were immunized intradermally with thefollowing preparations:

-   -   “purified protein derivative” (PPD)/tuberculin (WEYBRIDGE) 2 μg    -   purified 65 kDa M. leprae protein 50 μg and 100 μg

The diameter of the induration was measured 48 h after the injection ofthe different antigens on each of the 5 animals constituting the group.A positive induration greater than 8–10 mm is considered as positive.

It was observed that BCG erp::Kn induced a DTH after immunization withPPD and the 65 kD M. leprae protein.

EXAMPLE 6 Genotypic and Phenotypic Characterization of BCG erp::Kn

The characterization of the M. bovis mutant BCG erp::Kn was carried outin two ways. In a first instance, the genomic DNA of the mutant BCG (M)was extracted and analyzed by the Southern molecular hybridizationtechnique (Berthet et al., 1995) in comparison with the genomic DNA ofthe parental BCG strain (P). Digestion with EcoRI indicates that thegenome of BCG erp::Kn has lost such a site, located in the erp gene anddestroyed by the insertion of the kanamycin cassette. Furthermore,digestion with PstI, indicates that the restriction fragment carryingerp in BCG erp::Kn comprises an insert of 1.3 kbp corresponding to thepresence of the kanamycin cassette. These data confirm the replacementof the wild-type erp allele with a mutated erp::Kn allele in BCGerp::Kn.

In a second instance, the expression of the ERP protein was analyzed inthe wild-type BCG and in BCG erp::Kn by immunodetection according to theso-called “Western blot” method (Sambrook et al., 1989) with the aid ofan anti-ERP rabbit serum. The ERP protein is no longer detectable in thesupernatant of BCG erp::Kn.

EXAMPLE 7 Preparation of the Mycobacterium tuberculosis H37RV Strain:H37RV erp::Kn

The H37RV erp::Kn strain is derived from the reference Mycobacteriumtuberculosis strain. The strain has the characteristic feature of nolonger producing the protein corresponding to the erp gene. The erp genedetermines the synthesis of a repetitive exported protein located at thesurface of the Mycobacterium tuberculosis complex bacteria. This strainwas constructed during a homologous recombination experiment byreplacing the wild-type copy of the erp gene with a mutated copy, usingthe plasmid pIPX56 as described above. The mutated version of the erpgene contains an insertion of a cassette conferring resistance tokanamycin at the level of the EcoRI restriction site. Such a mutationabolishes the synthesis of the gene for a functional erp protein. One ofthe phenotypes associated with the introduction of this mutation is theloss of the capacity to persist in mice.

EXAMPLE 8 Characterization of the Product of the Mycobacteriumtuberculosis erp Gene

To characterize the product of the M. tuberculosis erp gene, therecombinant ERP protein was purified and overproduced. The protein wassynthesized in E. coli fused with 6 histidine residues (ERP-6His).ERP-6His forms cytoplasmic inclusion bodies and is then purified byimmobilization on nickel affinity chromatography under denaturingconditions. Renatured, soluble ERP-6His is analyzed by a two-dimensionalelectrophoresis gel (Laurent-Winter, 1997).

ERP-6His is separated into two species having the same molecular weight(36 kDa) but differing as regards their isoelectric point (pI). Thepredominant form has a pI of 5.3 which corresponds to that calculatedfor ERP-6His. The minor form is more acidic (pI 5.2), is likely tocorrespond to an aberrant form appearing in the cytoplasm of E. coli.This preparation of ERP-6His was used to immunize rabbits and apolyclonal serum with a high titer was obtained. Immunoreactive bands of36 and 34 kDa were detected by means of this serum both in the fractionsassociated with the cells and with the culture filtrates precipitatedwith TCA (trichloroacetic acid) of BCG and M. tuberculosis (strain Mt103). The larger band comigrated with recombinant ERP-6His. The 34 kDaband might be the result of a proteolytic degradation or alternativelyof a post-translational treatment taking place in M. tuberculosis. Thesedata are in agreement with those showing that the PGLTS (SEQ ID NO: 5)antigen, an M. bovis protein having more than 99% identity with the M.tuberculosis ERP protein, is present in the form of a doublet of similarmolecular weight in concentrated cellular fluids (BIGI et al., 1995).

On the basis of the structural characteristics, it has been suggestedthat the ERP protein may also be present at the surface of bacteria. Todetermine precisely the subcellular location of ERP, the attached M.tuberculosis bacillus was brought into contact with an anti-ERP serumand then incubated with a gold-labeled anti-rabbit conjugate.Observation by transmission electron microscopy revealed an intensesurface labeling at the periphery of the bacillus, indicating that ERPis a molecule exposed at the surface. This result was confirmed by theobservation of the labeled cell wall on section tubes of M. tuberculosis(data not shown).

It was then determined if ERP was produced during intracellularmultiplication of M. tuberculosis inside the cultured macrophages. Forthis purpose, J774 mouse macrophages were infected with a clinicalisolate of M. tuberculosis and were then observed by immunoelectronmicroscopy.

While no significant labeling was observed with the ERP preimmune serum,a specific labeling of the mycobacterial cell wall and of the phagosomallumen was observed with the serum of rabbit immunized with ERP.Furthermore, small vesicles containing labeled ERP were observed in theimmediate vicinity of the phagosomes. This demonstrates that ERP isproduced in the phagosomes of M. tuberculosis and suggests that ERPmoves around inside the cells.

EXAMPLE 9 Role of the ERP Protein in the Intracellular Growth ofMycobacteria

It was then examined if ERP was an essential bacteria component for theintracellular growth stage. For this purpose, a targeted null mutationwas introduced into the erp locus of the M. tuberculosis H37Rv strainand into the M. bovis BCG strain and into the model vaccine strain of M.bovis BCG. A suicide vector which is counterselectable with sucrose,pJQ200, was used to introduce a mutant allele of erp (erp::aph) into theM. bovis BCG chromosome.

The corresponding M. tuberculosis was constructed using the ts-sacBtechnology (Pelisic et al., 1997).

Mutant strains resulting from alleleic exchange were called BCG erp::aphand H37Rv erp::aph. A single copy of erp was reintroduced at the attBsite of BCG erp::aph and H37Rv erp::aph by means of the integrativevector derived from the mycobacteriophage MS6 (pAV6950). Analysis of thechromosomal DNA extracted from the parental strain (P), mutant strain(M) or complemented strain (complemented is understood to mean thereintroduction of a functional erp gene capable of directing thesynthesis of the ERP protein) revealed that the EcoRI site cut duringthe construction of erp::aph was also lost in the genome of the mutantand complemented strains. Furthermore, analysis using PstI indicates aninsertion of 1.3 kb inside the restriction fragment carrying erp.Hybridization of the same membrane with the sequences of the vectorspJQ200 and pPR27 did not make it possible to detect any signal (data notshown) suggesting that only the erp::aph cassette was introduced intothe genome of BCG erp::aph and H37Rv erp::aph. Analysis of the fractionsassociated with the cells and of the concentrated supernatants fromcultures of BCG erp::aph and H37Rv erp::aph indicated that theinterruption of erp had abolished the production of ERP. This wasconfirmed by immunoelectron microscopy by the disappearance of the goldlabeling on M. bovis BCG erp::aph cryosections. By contrast, theintegration of erp at the attB site of the mutant erp::aph strainsrestored the production of ERP both at the surface of the cells and inthe M. tuberculosis and M. bovis culture medium. Morphological analysisof the colony, doubling the length of time and the growthcharacteristics in the Middelbrook 7H9/ADC culture or the minimum Sautonmedium, did not make it possible to identify any difference between themutant, parental and complemented strains of BCG and H37Rv. Takentogether, these data show that erp is not essential for the growth ofBCG and H37Rv under laboratory conditions.

EXAMPLE 10

The capacity of BCG erp::aph and H37Rv erp::aph to grow in phagocyticcells was examined. For this purpose, the multiplication of the mutantand parental strains in a culture of macrophages derived from cellularmarrow was compared. As shown in FIG. 4A, the counting of the CFUsindicates that the erp::aph mutants do not multiply inside the mousemacrophages whereas the parental and complemented strains have a normalgrowth. Furthermore, the H37Rv erp::aph strain shows a reduction in thecytopathic effects compared with the parental and complemented strains(FIG. 4B). To determine if the erp::aph mutation also affectsmultiplication inside the host, the persistence of BCG erp::aph andH37Rv erp::aph in mice was analyzed. 106 viable units of the parentalstrains, mutant strains erp::aph and erp-complemented strains wereinjected by the intravenous route into Balb/c mice and the bacterialinfection was monitored by counting the CFUs after a period of 56 days(Lagranderie et al., 1996). The counting was carried out on the lungs,the liver and the spleen, three organs known to contain the highestmicrobacterial load after inoculation by the intravenous route. Asrepresented in FIG. 5A, the BCG erp::aph mutants were rapidly eliminatedfrom the lungs of the infected animals whereas the correspondingparental and complemented strains colonized this tissue and survived. Bycontrast, the H37Rv erp::aph mutant survived but multiplied very slowlycompared with the parental and complemented strains. The lungs representthe site of infection by the members of the M. tuberculosis complexduring tuberculosis. The multiplication of the erp::aph mutants was alsogreatly reduced in the liver (FIG. 5B) and the spleen (FIG. 5C).Furthermore, the morphology of the BCG colonies after having infected ananimal is very different: whereas the parental BCG gives rise to aso-called “diffuse” colony morphology, BCG erp::aph no longer diffusesand shows delayed growth (up to one week compared with the parentalstrain). The significance of this observation is unknown but the loss ofthe “diffuse” phenotype was correlated with the lowest levels ofresidual virulence among the BCG substrains (Dubos and Pierce, 1956,Pierce and Dubos, 1956, Pierce, Dubos and Scheiffer, 1956 and Dubos andPierce, 1956). The “nondiffuse” phenotype is not permanent and is lostafter restriction of the culture medium on 7H11. Furthermore, thereintroduction of erp restores the parental phenotype. Be that as itmay, these data demonstrate that the erp expression is required duringthe stage of intracellular growth of the mycobacteria belonging to theM. tuberculosis complex.

TABLE No. of sera 65 kDa Origin tested MBP-ERP* ERP-His6 BCG Humans  4 −− +/− monitored Humans (Bligny) 21 − − + Tuberculosis (In Pool)sufferers M. tuberculosis Humans (Uganda) 10 ND +++ (3/10) NDTuberculosis sufferers M. tuberculosis Children  4 − − + (Necker) (InPool) Tuberculosis sufferers M. tuberculosis (Direct Exam.+) Humans  6 −− + (Madagascar) Tuberculosis sufferers M. bovis Humans (Nepal)  1 Pool+++ +++ +++ Lepromatous leprosy sufferers M. leprae Bovins  4 +++ ++++++ Tuberculosis sufferers M. bovis *ERP protein fused with the MaltoseBinding Protein The serum of the individuals tested (or the pool in thecase of leprosy sufferers) was brought into contact with each of thethree proteins mentioned in the table. The immune response was measured.

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1. A Mycobacterium strain comprising a modified erp gene in its genome,wherein the strain is a strain selected from the complex ofMycobacterium tuberculosis species.
 2. The Mycobacterium strainaccording to claim 1, wherein the strain is recombinant.
 3. TheMycobacterium strain according to claim 1 or 2, wherein the erp gene ismodified by mutation, insertion, deletion, or substitution.
 4. TheMycobacterium strain according to claim 3, wherein the mutation,insertion, deletion, or substitution is carried out on at least two basepairs.
 5. The Mycobacterium strain according to claim 3, wherein the erpgene is modified by insertion of a nucleotide or polynucleotide.
 6. TheMycobacterium strain according to claim 5, wherein the insertedpolynucleotide comprises a selectable gene.
 7. The Mycobacterium strainaccording to claim 6, wherein the selectable gene imparts resistance toan antibiotic.
 8. The Mycobacterium strain according to claim 7, whereinthe antibiotic is kanamycin, spectinomycin, or hygromycin.
 9. TheMycobacterium strain according to claim 1, wherein the strain is capableof expressing a polynucleotide encoding an antigen of a Mycobacterium ofa species other than that to which said strain belongs.
 10. TheMycobacterium strain according to claim 1, wherein the strain is capableof expressing a polynucleotide foreign to the Mycobacterium genus.
 11. AMycobacterium strain according to claim 3, wherein the erp gene lacksits repeat sequences.
 12. An immunogenic composition comprising aMycobacterium strain according to claim 1, wherein the composition is ina form suitable for administration to a human or an animal, and whereinthe Mycobacterium strains is presented in an amount sufficient to inducean inmune response in the human or animal when the composition isadminitered.
 13. A method for the production of a Mycobacterium strainwith an erp gene in its genome, wherein the erp gene is modified, andwherein the strain is a strain selected from the complex ofMycobacterium tuberculosis species, comprising: transforming, with avector comprising a modified erp gene, a Mycobacterium strain of thecomplex of Mycobacterium tuberculosis species propagated at a permissivetemperature, culturing colonies resulting from the transformation on amedium supplemented with a selectable product and sucrose, and isolatingthe strain thus selected.
 14. The method according to claim 13, whereinthe selectable product is an antibiotic chosen from kanamycin,spectinomycin and hygromycin.
 15. An immunogenic composition comprisinga Mycobacterium strain obtained using the method according to claim 14.16. The immunogenic composition of claim 15, further comprising at leastone pharmaceutically compatible excipient.
 17. An immunogeniccomposition comprising a Mycobacterium strain obtained using the methodaccording to claim
 13. 18. The immunogenic composition of claim 17,further comprising at least one pharmaceutically compatible excipient.19. A method of screening individuals, to whom an immunogeniccomposition according to claim 16 or claim 18 has been administered,comprising detecting an absence, from a biological sample from saidindividuals, of antibodies directed against a whole or part of apurified ERP protein.
 20. A method of screening individuals, to whom animmunogenic composition according to claim 16 or claim 18 as beenadministered, comprising detecting, in a biological sample from saidindividuals, T cells directed against a whole or part of a purified ERPprotein.
 21. A method of preparing a Mycobacterium strain according toclaim 1, comprising: providing a recombinant vector comprising amodified erp gene, and combining said recombinant vector with aMycobacterium strain of the complex of Mycobacterium tuberculosisspecies comprising an erp gene, under conditions where allelic exchangecan occur, wherein allelic exchange results in replacement of said erpgene with said recombinant vector.